1. Field of the Invention
The concept underlying the gist of the instant invention is based on the unexpected discovery that two classes of N-Halamine compounds (N,N'-DIHALO-2-IMIDAZOLIDINONES and N-HALO-2-OXAZOLIDINONES) are effective urease and nitrification inhibitors. Members of the N,N'-DIHALO-2-IMIDAZOLIDINONE class of compounds have been previously patented in U.S. Pat. No. 4,681,948, Worley, July 21, 1987, who also described the use of this class of compounds as biocides for control and prevention of microorganisms in aqueous media, particularly industrial water systems, potable water, swimming pools, hot tubs and waste water treatment facilities, and in sanitizing applications. Similarly, members of the N-HALO-2-OXAZOLIDINONE class of compounds have been patented in U.S. Pat. Nos. 3,591,601, Walles, July 6, 1971; 3,931,213, Kaminski et al., Jan. 6, 1976; and 4,009,178, Bodor et al., Feb. 22, 1977; and their use as biocides described. U.S. Pat. No. 4,000,293, Kaminski et al., Dec. 28, 1976, teaches the use of 3-chloro-2-oxazolidinones for inhibiting bacterial growth. More recently the 3-chloro2-oxazolidinones have been patented for controlling the growth of the microorganism Legionella pneumophila in recirculating aqueous mediums in air cooling systems U.S. Pat. No. 4,659,484, Worley et al., Apr. 21, 1987. Even though the N,N'-DIHALO-2 IMIDAZOLIDINONES and N-HALO-2-OXAZOLIDINONES have been previously patented, supra, their use as inhibitors of urease catalyzed hydrolysis of urea in aqueous and soil systems and as inhibitors of the nitrification of ammonium nitrogen in soil systems have heretofore been unknown.
2. Description of Prior Art
Embodiment 1: Although several hundred scientific papers have been published on ureas since Summer 1926) first produced the classical octahedral crystals and showed that the enzyme was a protein, it was not until 1969 that Zerner's group [R. L. Blakeley, E. C. Webb, and B. Zerner, Biochemistry 8, 1984-1990 (1969)] first prepared a highly purified urease with a full specific activity and in at least a 99% homogeneous state. They established with this preparation a reproducible molecular weight (about 590,000) and proposed that the molecule contained six subunits with asparagine as the N-terminal amino acid. Although previous work (J. F. Ambrose, G. B. Kistiakowsky, and A. G. Kridl, J. Am. Chem. Soc. 73, 1232 (1951)] had indicated that four or eight essential SH-groups were involved in the urea-hydrolysis reaction, Zerner's group confirmed that the active site SH-groups "react slowly with N-ethylmaleimide," but they were unable to define unequivocally the number of "essential SH groups" in the 590,000 molecular weight species. In addition, Kobashi et al. [K. Kobashi, J. Hase, and T. Komai, Biochem. Biophys. Res. Commun. 23, 34 (1966)], on the basis of inhibition by hydroxamic acids, suggested that the number of active sites in the 590,000 molecular weight species of sword bean urease was two. These results seem to be confirmed by the discovery that highly purified urease from Jack Bean [N. E. Dixon, C. Gazzola, R. L. Blakeley, and B. Zerner, J. Am. Chem. Soc. 97 4131 (1975)] and from tobacco, rice, and soybean [J. C. Polacco, Plant Science Letters 10 249-255 (1977)] contained stoichiometric amounts of nickel (two atoms per active site), demonstrating simultaneously the first biological role definitely assigned to nickel.
Over the last few years considerable effort has been made to elucidate the mechanism of the urease reaction. Elucidation of the mechanism of the urease reaction is complicated because the enzyme has a tendency to form polymers and isozymes changing the properties of the original monomeric enzyme and probably the mechanism of reaction [W. N. Fishbein and K. Nagarajan, Arch. Biochem. Biophys. 144, 700-714 (1971)]. In addition, the properties of soil urease differ significantly from those of ureases from other sources [J. M. Bremner and R. L. Mulvaney, Soil Enzymes, R. G. Burns, editor, Academic Press, 149-196 (1978)]; it is much more difficult to obtain reliable kinetic data for enzymes in heterogeneous environments, such as soil, than for enzymes in homogeneous solutions. As a result, most attempts to elucidate the mechanism of urease inhibition have been confined to nonsoil systems.
Attempts to demonstrate the formation of a carbamoyl-enzyme intermediate, which was postulated many years ago, have so far failed; Zerner's group [N. E. Dixon, P. W. Riddles, C. Gazzola, R. L. Blakeley, and B. Zerner, Can J. Biochem. 58, 1335-1344 (1980)] proposed a mechanism of reaction on the basis of a carbamoyl-transfer reaction and where the substrate is activated toward nucleophilic attack by O-coordination to a Ni.sup.+2 ion. Both Ni.sup.+2 ions are involved in this proposed mechanism. A second mechanism of reaction based on the determination of kinetic isotope effects [R. Medina, T. Olleros, and H. L. Schmidt, Proc. 4th Int. Conference on Stable Isotopes, H. L. Schmidt, H. Forstel, and K. Heizinger, editors, Julich, Mar. 1981, Elsevier, Amsterdam, 77-82 (1982)] was proposed. These results indicated the existence of an enzyme-bound carbamate intermediate and demonstrated that the enzyme-Ni-substrate complex decomposes, releasing the first NH3 in a slow, rate-limiting step.
Most enzymes can be poisoned or inhibited by certain chemical reagents. There are two major types of enzyme inhibitors: irreversible and reversible. Irreversible inhibitors combine with or destroy a functional group on the enzyme molecule that is necessary for its catalytic activity. Reversible inhibitors generally are considered to be either competitive or noncompetitive. Competitive inhibitors compete with the substrate for binding to the active site, but once bound cannot be transformed by the enzyme. An identifying feature of competitive inhibition is that it can be reversed by increasing the substrate concentration. Noncompetitive inhibitors do not bind at the site on the enzyme at which the substrate does; however, their binding to the enzyme alters the structure or conformation of the enzyme so that reversible inactivation of the catalytic site results.
Many urease inhibitors have been identified; however, few kinetic descriptions include the type of inhibition. The reversible and competitive inhibition of sword bean urease by a wide variety of hydroxamic acids was discovered by Kobashi et al. [K. Kobashi, J. Hase, and K. Uehara, Biochim. Biophys. Acta 65, 380-383 (1962)]. Kinetic and spectral studies performed by B. Zerner and coworkers [N. E. Dixon, J. A. Hinds, A. K. Fihelly, C. Gazzola, D. J. Winzor, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58, 1323-1334 (1980)] established that hydroxamic acids were reversibly bound to active-site nickel ions in Jack Bean urease. Chemical and Physical studies of the enzymatically inactive phosphoramidate-urease complex provide convincing evidence that Phosphoramidate binds reversibly to the active-site nickel ion [N. E. Dixon, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58, 481-488 (1980)].
The kinetics of urease inhibition by phenyl phosphorodiamidate (which demonstrates a competitive inhibition) and hydroquinone (which exemplifies a mixed inhibition mechanism) were performed by L. J. Youngdahl and E. R. Austin at the International Fertilizer Development Center (IFDC, unpublished results). A kinetic study of the soil urease inhibition by six substituted ureas, which are used as herbicides, showed that all six compounds exhibited mixed inhibition characteristics (competitive and noncompetitive) [S. Cervelli, P. Nannipieri, G. Giovannini, and A. Perna, Pesticide Biochem. Physiol. 5, 221-225 (1975)].
Many other compounds have been identified as urease inhibitors. Mulvaney and Bremner [R. L. Mulvaney and J. M. Bremner, Soil Biochem. 5, 153-196 (1981)] have published an extensive review on urease inhibitors. More recently, Gould and coworkers [W. D. Gould, C. Hagedorn, and R.G.L. McCready, "Urea Transformations and Fertilizer Efficiency in Soil," Advances in Agronomy 40, 209-238 (1986)] described a number of urease inhibitors. These inhibitors were classified into three groups (sufhydryl reagents, hydroxamates, and structural analogs of urea and related compounds). Members of these three classes of compounds plus a fourth class (agricultural chemicals which inhibit urease) have been more extensively described by Medina and Radel (R. Medina and R. J. Radel, "Mechanisms of Urease Inhibition," submitted for publication in Ammonia Volatilization From Urea Fertillzers, Bert R. Bock and David E. Kissel, editors, to be published by the Tennessee Valley Authority).
Although a wide variety of chemicals have been shown to inhibit urease activity, none have yet been commericalized. Phenyl phosphorodiamidate (PPDA), (C6H5O)PO(NH2)2, has probably been the most widely studied urease inhibitor in recent years and until recently was considered the best known inhibitor. Extensive work directed at commercializing PPDA has been conducted at the Tennessee Valley Authority's National Fertilizer Development Center. Most of this work has been directed at determining the compatibility of PPDA at process conditions typically encountered during urea granulation [J. Gautney, Y. K. Kim, and P. M. Gagen, I&EC Prod. R&D 23, No. 3, 483-489 (1984)], in fluid fertilizers [J. Gautney. Y. K. Kim, and A. R. Barnard, "Solubilities and Stabilities of the Nitrogen Loss Inhibitors Dicyandiamide, Thiourea, and Phenyl Phosphorodiamidate in Fluid Fertilizers," I&EC Prod. R&D 24, No. 1, 155-161 (1985)], and in solid urea during long-term bulk storage [J. Gautney, A. R. Barnard, D. B. Penney, and Y. K. Kim, "Solid-State Decomposition Kinetics of Phenyl Phosphorodiamidate," Soil Science Society of America Journal 50, 792-797 (1986)]. The results of these studies clearly indicate that PPDA can be cogranulated with urea but decomposes relatively rapidly in solid mixtures with urea and in fluid fertilizers. As a result, commercialization of PPDA does not, at this time, appear to be promising.
Two relatively new inhibitors, thiophoshoryl triamide, (TPTA), (NH2)3PS and N-(n-butyl)-thiophosphoryl triamide, (NBTPTA), [NH(CH3CH2CH2CH2)] (NH2)2PS, are currently receiving considerable attention in terms of commercial development. Both of these compounds have been shown to yield inhibition characteristics which are superior to those of PPDA. The Tennessee Valley Authority has been granted a statuatory invention registration on TPTA (SIR No. H25, Radel, Feb. 4, 1986) and U.S. Pat. No. 4,676,822, Gautney, June 30, 1987, on the use of TPTA in fluid fertilizers. The inhibitor NBTPT was patented by Allied Corporation in U.S. Pat. No. 4,530,714, Kolc et al., July 23, 1985.
Fertilizer compatibility studies with TPTA (TVA Bulletin Y-191, "New Developments in Fertilizer Technology," October 1985) showed that this inhibitor can be cogranulated with urea and is relatively stable in fluid fertilizers containing urea, but like PPDA, decomposes in solid mixtures with urea (R. J. Radel, J. Gautney, A. A. Randle, J. E. Cochran, R. M. Miles, H. M. Williams, B. R. Bock, and N. K. Savant, "Evaluation of Thiophosphoryl Triamide as a Urease Inhibitor," paper presented at the 194th National Meeting of the American Chemical Society, Aug. 30-Sep. 4, 1987, New Orleans, LA). As a result, TPTA has considerable potential for use in urea containing fluid fertilizer but limited potential for use with solid urea. No data are currently available on the compatibility of NBTPTA with fertilizer materials. Field studies with TPTA and NBTPTA in urea containing fluid fertilizers are in progress.
Even though urease has been studied extensively for about 60 years and a large number of compounds have been identified as potential urease inhibitors, the fact that no such urease inhibitor has been commercially developed indicates the need to find, identify, and/or develop new and better compounds for such purposes. The invention described herein covers the use of two classes of N-halamine compounds as urease inhibitors. These compounds were not previously known to inhibit urease activity.
Embodiment 2: The biological oxidation (nitrification) of ammonium nitrogen (NH4.sup.+) to nitrate nitrogen (NO3.sup.-) reaction (3), supra, is an energy producing process involving a loss of eight electrons by the ammonium nitrogen and resulting in a nitrogen valence change from -3 to +5. The process is carried out in soils mainly by chemosynthetic autotrophs (Nitrobacteriaceae) in order to derive energy needed for their metabolic activities. The probable reaction sequence for the first step of nitrification (conversion of ammonium to nitrite by nitrosomonas) is: ammonia----hydroxylamine-----(nitroxyl?)-----(nitrohydroxylamine)?-----nit rite. The postulated intermediates nitroxyl (NOH) and nitrohydroxylamine (NO2.cndot.NH2OH) have not been positively identified, but their participation in the reaction sequence is consistent with the assumption that two electrons are involved in each intermediate oxidation step from ammonium to nitrite (R. D. Hauck, "Mode of Action of Nitrification Inhibitors," Nitrification Inhibitors-Potentials and Limitations, ASA Special Publication No. 38, 1980, American Society of Agronomy, Madison, WI). The second step of nitrification (conversion of nitrite to nitrate) is a single step process which is carried out mainly by nitrobacter.
Numerous extensive and in-depth searches have been conducted during the past 25 years to find or identify chemicals that can inhibit the biological oxidation of ammonium to nitrate in soils. Since nitrite can be phytotoxic to plants, it is desirable that the first step of the nitrification process, the conversion of ammonium to nitrite, be preferentially inhibited because this avoids subsequent buildup of nitrite. A large number of chemicals have been found to inhibit nitrification. These chemicals include pyridines, pyrimidines, mercapto compounds, succinamides, thiazoles, triazoles, triazines, cyanamid derivatives, and various thio compounds [R. D. Hauck, "Technological Approaches to Improving the Efficiency of Nitrogen Fertilizer Use by Crop Plants," Nitrogen in Crop Production, American Society of Agronomy, Madison WI, 552-560 (1984)]. A number of agricultural pesticides also have been shown to inhibit nitrification. The herbicides N-N'-dimethyl-4-4-chlorophenyl urea, and ethyl and isopropyl carbamate have been shown to inhibit nitrification [M. H. Briggs and I. Segal, Life Science 2, 69-72 (1963)]. The pesticides diazion, manozeb, benzene hexachloride, pentachlorophenol, Vapam, maneb, and isodoacetic acid are also known nitrification inhibitors [T. Nishihara, Bull. Fac. Agr. Kagoshima Univ. 12, 107-158 (1962)].
In general, most nitrification inhibitors function by either retarding the growth and/or other life support function of nitrifiers. Inhibition can result from interference with the nitrifiers respiration and cytochrome oxidase function, by production of acid in the microenvironment, by chelation of essential metal ions, and by liberation of toxic compounds such as mercaptans, sulfoxides, and sulfones.
The biochemistry of several naturally occurring and synthetic substances which can act as nitrification inhibitors have been reviewed [J. H. Quastel, Ann. Rev. Plant Physiol. 16, 217-240 (1965)]. Methionine and some alkylmercapto amino acids delay the beginning of nitrification. This delay is believed to result from retardation of the proliferation of nitrifying organisms. Cystine and cysteine indirectly impede nitrification. Quastel [J. H. Quastel, Plant Physiol. 3, F. C. Steward, editor, Academic Press, New York, 671-756 1963)] suggested that these compounds are decomposed by soil heterotrophs with the formation of sulfuric acid which decreases soil pH to a level less favorable for nitrification. Later, researchers [J. M. Bremner and L. G. Bundy, Soil. Biol. Biochem. 6, 161-165 (1974)] presented evidence that cystine, cysteine, methionine, and other nonvolatile organic sulfur compounds may inhibit nitrification by decomposition to toxic carbon disulfide. Thiourea and allylthiourea are thought to inhibit nitrification by complexing with metallic cations such as Cu.sup.+2, which are needed for the process of nitrification [J. H. Quastel, Ann. Rev. Plant Physiol. 16, 217-240 (1965)]. Copper chelating agents such as salicyladoxime and sodium diethyldithiocarbamate also retard nitrification [H. Lees, Metabolic Inhibitors 2, R. H. Hochester and J. H. Quastel, editors, Academic Press, New York, 615-629 (1963)]. Addition of copper cation (Cu.sup.+2) has been shown to somewhat counteract the nitrification inhibition effect of thiourea but not that of allylthiourea [E. R. Campbell and M.I.H. Aleem, Antione van Leeuwenhoek, J. Microbiol. Serol. 31, 124-136 (1965)]. In contrast to thiourea, the inhibitors ethyl urethane [J. H. Quastel, Plant Physiol. 3, F. C. Steward, editor, Academic Press, New York. 671-756 (1963)], 2-chloro-6-(trichloromethyl) pyridine [C.A.I. Goring, Soil Science 93, 211-218 (1962)], sodium azide, and dicyandiamide retard nitrification by acting directly on the nitrosomonas species involved in the first step of nitrification [T. Nishihara, Bull. Fac. Agr. Kagoshima Univ. 12, 107-158 (1962)]. The inhibitors phenyl mercuric acetate, mono- and di-chlorophenyl isothiocyanates, and sodium chlorate preferentially inhibit the second step of the nitrification process, the conversion of nitrite to nitrate, [R. D. Hauck, "Synthetic Slow Release Fertilizers and Fertilizer Amendments," Organic Chemicals in the Soil Environment 2, C.A.I. Goring and J. W. Hamaker, editors, Marcel Dekker, New York, 633-690 (1972)].
Although a large number of many different types of compounds have been found to inhibit nitrification (many of which are patented), only seven have been recently produced commercially for use as nitrification inhibitors. Four of these [2-amino-4-chloro-6-methyl pyridine (AM), 2-mercaptobenzothiazole (MBT), sulfathiazole (ST), and thiourea (TU) are produced and marketed primarily in Japan.
In the U.S. the compound 2-chloro-6-trichloromethyl pyridine (nitrapyrin) was commercialized as N-Serve in 1974 and has remained on the market since that time. Another compound, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole (ETT), was licensed in the U.S. as a nitrification inhibitor by Olin Corporation in 1982. Olin had previously marketed the compound as a fungicide under the trade name Terrazole. Terrazole was marketed by Olin as a nitrification inhibitor under the trade name Dwell for about one year but was withdrawn from the market at about the same time that Olin's Agrichemicals Division was purchased by Uniroyal Inc., on Oct. 1, 1983. Apparently, Uniroyal is not presently marketing Dwell.
The compound dicyandiamide has received much attention in the U.S. in recent years because it offers some distinct advantages over nitrapyrin and ETT. Both nitrapyrin and ETT have relatively high vapor pressures. These vapor pressures prevent cogranulation of nitrapyrin and ETT with solid fertilizers such as urea. Their use with nitrogen solutions is also limited because of volatilization losses during fertilizer processing and application. As a result, nitrapyrin and ETT are used primarily with anhydrous ammonia; however, even then, special precautions must be taken. Nitrapyrin can be tank mixed with anhydrous ammonia but has a maximum recommended shelf life of three weeks (Dow Chemical Company USA, "N-Serve Nitrogen Stabilizer," Technical Information Bulletin, Ag-Organics Department, Midland, MI). ETT cannot be tank mixed with anhydrous ammonia and therefore must be applied simultaneously from a separate tank.
Dicyandiamide, or the other hand, has very little vapor pressure and as a result can be cogranulated with solid fertilizers such as urea without significant losses of inhibitor [J. Gautney, Y. K. Kim, and P. M. Gagen, Ind. Eng. Chem. Prod. Res. Dev. 23, No.3, 483-489 (1984)]. Dicyandiamide is also stable in anhydrous and aqueous ammonia solutions [J. Gautney, Y. K. Kim, and P. M. Gagen, Ind. Eng. Chem. Prod. Res. Dev. 24, No.4, 645-650 (1985)].
Dicyandiamide is produced and marketed in Japan and more recently is being produced in West Germany and test marketed in the U.S. as a slow release nitrogen fertilizer. Dicyandiamide currently is not registered as a nitrification inhibitor in the U.S.. Although dicyandiamide offers a number of advantages over nitrapyrin and ETT in terms of fertilizer compatibility, like nitrapyrin and ETT, it is relatively expensive for use with fertilizer materials.
From the above information it is clear that although a large number of compounds have been identified and/or patented as nitrification inhibitors, there is no single inhibitor which meets all the needs of American agriculture. Thus, there is a need to find, identify, and/or develop new improved compounds for use as nitrification inhibitors.